The morphologies of cells, organs and organisms are defined by the ability of cells to develop and sense mechanical forces. Cancers, genetic malformations, and other diseases involve alteration of either force production, force sensing or rigidity sensing. Our long-range goal is to develop a quantitative, molecular understanding of the biochemical and biophysical functions underlying the processes of cell adhesion, migration and force generation, including the sensing of substrate rigidity. Recent findings have provided insight into rigidity sensing and the roles of different myosin II isoforms in periodic contractions. In the case of rigidity sensing, it is notably altered in many cancerous cells. In physical terms, rigidity is defined by the displacement per unit force and we suggest that both parameters are sensed at the leading edges of active lamellipodia. Our studies have identified several proteins that are required for sensing the rigidity of fibronectin matrices, avB3 integrin, RPTPa , Fyn, and p130Cas. In active lamellipodia, these proteins are concentrated near the leading edge and are linked enzymatically. Our working hypothesis is that force-dependent unfolding of p130Cas results in Fyn phosphorylation on rigid surfaces but on soft surfaces Fyn and p130Cas are mechanically displaced thereby inhibiting phosphorylation. We propose to determine if and how Fyn is immobilized at the leading edge during rigidity sensing. We will examine if p130Cas is stretched at the leading edge by a FRET assay and will study the j mechanism of its binding during rigidity sensing. In some cases, cells use periodic contractions to sense rigidity. Similar contractions are observed in many types of spreading and migrating cells. However, the roles of myosin II-A and II-B are dramatically different. The periodic nature of the contractions makes it easier to correlate assembly of the myosin filaments with the quantification of both vertical and lateral force generation in the fluorescent microscope. Vertical bending of the lamellipodium during normal contractions has important implications for 3-D motility and matrix remodeling. Control of the contractions depends upon myosin light chain kinase (MLCK) and possibly its transport on actin. Using GFP-myosin II-A or II-B, inhibitors and myosin-depleted cell lines, we will analyze whether localized assembly and phosphorylation correlate with force production. We will determine which MLCK domain is needed for localization and myosin activation. These quantitative analyses will provide an understanding of the biochemical and physical aspects of rigidity sensing and periodic contractions at a molecular level that can then be used for modeling and design of novel therapies for wound healing, metastasis, tissue malformation and functional tissue engineering.